Wild-type 4.5S RNA and sequence of the tetraloop mutants. Secondary structure of the wild-type 4.5S RNA with tetraloop sequence GGAA. Non-Watson–Crick base pairs are indicated by (•). The Ffh binding site indicates the two conserved internal loops. Domain IV is the universally conserved region of SRP RNA. Gray oval highlights the conserved GNRA tetraloop of 4.5S RNA.

GTPase activites of SRP–FtsY complex. Fixed concentrations of SRP or Ffh with varying amounts of FtsY (amino acids 47–498) in the presence of 100 μM GTP and trace amount of γ-³²P-GTP were used in this assay. The concentrations of wild type (•), CUUC (▪), GAAA (∇), and GUAA (▴) SRP were 0.2 μM whereas the concentrations of UUCG (Δ) and UUUU (□) SRP were 2 μM. In the absence of RNA (○), only 1 μM of Ffh was used. The extrapolated maximal rate constants for wild type, CUUC, GAAA, GUAA, UUCG, UUUU, and no RNA are 13.4 ± 4.1, 15.2 ± 5.1, 10.9 ± 2.8, 8.4 ± 2.5, 3.2 ± 1.4, 1.3 ± 0.4, and 2.2 ± 1.5 min⁻¹, respectively. The maximal rate constants and values of the data points on the curves are averages of at least three independent experiments.

ELISA of SRP–FtsY complex. SRP–FtsY complex formation with wild-type (•) and UUCG (Δ) 4.5S RNA was detected by ELISA using 50 nM of wild-type or UUCG SRP, 0.5 mM GMPPNP, and titrating concentrations of FtsY (amino acids 47–497). scFv IA02, which recognizes only the complex, was used to determine the amount of complex formed, and scFv IB11 was used to detect the total amount of SRP in each binding reaction. These data are representatives of three independent experiments.

Model of the SRP–FtsY complex and hydroxyl radical probing of full-length wild type, UUCG, and GUAA 4.5S RNA in a saturable SRP–FtsY (amino acids 197–498) complex derivatized by BABE-Fe at positions 344 of Ffh. (A) Model of the SRP–FtsY complex based on previous hydroxyl radical probing data (). G, N, and M indicate the GTPase, N-terminal, and M domains for Ffh and FtsY. FtsY is shown in green. NG and M domains of Ffh are shown in blue and purple, respectively. 4.5S RNA is shown in pink and the nucleotides of tetraloop is depicted in orange. Residues A359 and E244 of FtsY and E153 and M344 of Ffh are positions of BABE-Fe. (B) Lanes labeled −OH, T1, 1, and 2 indicate 4.5S RNA alkaline hydrolysis, partial T1 RNase guanosine cleavage, 4.5S RNA alone in buffer, or in the presence of cleaving reagents, respectively. All remaining lanes are hydroxyl radical probing reactions performed in the presence of GMPPCP and FtsY. +R and −R indicate the presence and absence of cleaving reagents. Numbers on the left indicate nucleotide positions of 4.5S RNA and bars indicate regions of cleavage sites. Quantitations of specific nucleotides are shown with normalization for loading differences between the +G and −G lanes for the three RNA constructs. Fold change indicates the intensity of the nucleotide in the +G reaction relative to the −G reaction. These data are representatives of three independent experiments.

Tethered hydroxyl radical probing of SRP–FtsY complexes. Strand scission of full-length wild-type (WT), UUCG, and GUAA 4.5S RNA in a saturable SRP–FtsY complex derivatized by BABE-Fe at positions Ffh E153 (A), FtsY A359 (B), and FtsY E244 (C). Lanes labeled −OH, T1, 1, and 2 indicate 4.5S RNA alkaline hydrolysis, partial T1 RNase guanosine cleavage, 4.5S RNA alone in buffer, or in the presence of cleaving reagents, respectively. Lanes labeled −G and +G indicate the absence and presence of GMPPCP, respectively. Nucleotide positions of 4.5S RNA are indicated on the left. Regions of cleavage sites for wild type and equivalent sites for UUCG and GUAA are indicated by bars. Quantitations of specific nucleotides are shown with normalization for loading differences between the +G and −G lanes for the three RNA constructs. Fold change indicates the intensity of the nucleotide in the +G reaction relative to the −G reaction. These data are representatives of three independent experiments.

In vivo complementation with 4.5S RNA mutants. E. coli strain S1610 transformed with plasmids encoding wild-type (•),UUCG (Δ), CUUC (▪), UUUU (□), GUAA (▾), and GAAA (∇) 4.5S RNA were grown at (A) 30°C and (B) 42°C. E. coli transformed with the specified 4.5S RNA construct was streaked onto an agar plate with ampicillin at 30°C. Liquid cultures of the specified 4.5S RNA constructs grown at 30°C were streaked onto an agar plate with ampicillin at 42°C. Growth of the E. coli harboring wild-type, UUCG, UUUU, CUUC, GUAA, and GAAA 4.5S RNA in LB media with ampicillin was monitored over time at the designated temperatures. No data were available for the UUCG mutant at 42°C since there was no growth observed at 30°C. These data are representatives of three independent experiments.

Model of the mechanism of 4.5S RNA in E. coli SRP-dependent protein translocation. Step 1, cytoplasmic SRP bound with GTP is loaded with a RNC containing a signal peptide. Step 2, the RNC is translocated to the inner membrane by SRP binding to its receptor (FtsY) bound with GTP. Step 3, interaction of SRP with FtsY induces a conformational rearrangement in the SRP as previously observed (). Presence of the signal peptide in the M domain perturbs interaction of the tetraloop of 4.5S RNA and the Ffh–FtsY heterodimer (indicated by the box) and inhibits GTP hydrolysis. Step 4, signal peptide release from the SRP and docking of the RNC with translocon. Step 5, tetraloop of 4.5S RNA interacts with the G domains of Ffh and FtsY and stimulates GTP hydrolysis of the SRP–FtsY complex and dissociation of SRP and FtsY. Tetraloop mutants (black oval) inhibit GTP hydrolysis and prevent recycling of SRP and FtsY. Step 6, exchange of GDP to GTP for SRP and FtsY and primed for a new round of protein translocation.